Biocompatible implant for the expression and in vivo secretion of a therapeutic substance

ABSTRACT

The invention relates to an implant obtained by assembling in vitro various elements in order to form a neo-organ which is introduced preferably in the peritoneal cavity of the recipient. The implant comprises a biocompatible support intended to the biological anchoring of cells; cells having the capacity of expressing and secreting naturally or after recombination a predetermined compound, for example a compound having a therapeutical interest; and a constituent capable of inducing and/or promoting the geling of said cells. The invention also relates to a kit for the preparation of the implant as well as to a new recombinant retroviral vector comprising a provirus DNA sequence modified in that the genes gag, pol and env have been deleted at least partially so as to obtain a proviral DNA capable of replication. The invention also relates to recombinant cells comprising the new retroviral vector.

[0001] The invention relates to a biocompatible implant for theexpression of defined substances in man or in animals, in particular forthe anchoring of the recombinant cells at the invention.

[0002] The present invention also relates to retroviral vectors for thepreparation of recombinant cells capable of being implanted in vivo fora therapeutic purpose

STATE OF THE ART

[0003] The in vivo introduction of implants capable of expressingdefined substances for example for therapeutic purposes necessitates theuse of efficacious agents as regards the desired therapeutic orprophylactic objective and that the organism into which the implant isintroduced has the capacity to tolerate it in the relatively long-term.

[0004] The earlier international patent application published under thenumber 92/15676 described agents to obtain in vivo the expression ofdefined nucleotide sequences for the purpose of a therapeutic treatmentof diseases resulting from a genetic anomaly. This internationalapplication 92/15676 proposes the use of fibroblasts geneticallymodified by a retroviral vector for the purpose of implanting them inthe connective tissue of the skin of the subject to be treated.

[0005] The nucleotide sequence whose expression is desired in thisearlier international application is paced under the control of the LTR(Long Terminal Repeat) sequence of the retroviral vector and/or underthe control of inducible or constitutive exogenous promoters, the LTRsequence of the retravirus being nonetheless conserved when theexogenous promoter is present.

SUMMARY OF THE INVENTION

[0006] The invention proposes agents making it possible to achieve invivo the expression of a selected nucleotide sequence under conditionssuch that this expression is obtained aver a period of several months,preferably more than 6 months, in a manner such that the product of thenucleotide sequence expressed is present in sufficient quantity andunder conditions suitable for the production of a desired therapeuticeffect in vivo.

[0007] The agents defined in the present application make it possible toachieve the expression and the secretion of a protein, glyco-protein ora peptide of biological interest to justify its therapeutic use in vivo.

[0008] The invention also relates to the use of biocompatible materialsas supports for the introduction into man or animals of cells forexample recombinant cells of the invention defined below, starting fromwhich it is desired to produce a defined molecule in particular fortherapeutic purposes.

[0009] The preparation of implants requires such supports suitable forbeing placed in contact with cells and various factors promoting theadhesion of these cells to the support if necessary, under conditionssuch that the different constituents present conserve their principalnatural structural and functional properties.

[0010] These biocompatible supports whether of biological origin or notmay or may not be resorbable by the host into which they are introduced.

[0011] The invention also concerns retroviral vectors having thecapacity to infect cells, and in particular eukaryotic cells, as well asthe recombinant cells including these vectors. The recombinant cells ofthe invention may be administered or implanted in a patient, thusproducing in vivo a protein or any expression product of a definednucleotide sequence inserted into the retroviral vector.

[0012] The inventors have examined which are the parameters which, atvarious levels, would make it possible to express in vivo a definednucleotide sequence so as to improve the expression of the nucleotidesequence inserted in the vector in order to obtain a therapeutic effect,if necessary of long duration.

[0013] Thus, they have demonstrated that when the exogenous nucleotidesequence whose expression is desired is advantageously placed in thevector intended for the infection of the cells under the control of aninducible or constitutive exogenous promoter, the LTR sequence internalto the retroviral vector must then be partially deleted. The deletionmust be sufficient to impair the transcription of the mRNA

DETAILED DESCRIPTION OF THE INVENTION Biocompatible Implant

[0014] The implant or neo-organ of the invention is obtained by theprocess which insists in the in vitro assembly of various elements inorder to form an implant which is introduced preferably into theperitoneal cavity of the recipient. Other implantation sites can be usedsuch as the perirenal space, the skin. In vivo the implant gives rise toa connective tissue formed de novo which is vascularized and unmodifiedover the course of time.

[0015] In general, the implant of the invention is characterized in thatit comprises:

[0016] a biocompatible support making possible the biological anchoringof cells,

[0017] cells having the capacity to express and secrete naturally orafter recombination a defined substance, for example, a substance oftherapeutic interest, and

[0018] a constituent capable of inducing and/or promoting the gelationof said cells.

[0019] In particular, the elements participating in the assembly of thein vitro implant are the following:

[0020] 1) A rigid support. It may be prepared from differentbiomaterials: PTFE coral, cross-linked collagen fibers;

[0021] 2) A collagen gel. It is possible to use rat tail collagen,bovine collagen, human collagen; and

[0022] 3) Genetically modified cell, preferably the recombinant cell'sof the present invention which are described in detail below.

[0023] Hence the object of the invention is an implant or neo-organcharacterized in that it comprises a biocompatible rigid support, inparticular a rigid support made of PFTE or of biological origin, makingpossible the biological anchoring of cells previously placed or notplaced in contact with constituents capable of inducing and/or promotingtheir inclusion within a gel-forming matrix, said cells being chosen fortheir capacity to express and secrete naturally or after recombination adefined substance, for example a substance of therapeutic interest.

[0024] The expression “biological anchoring” means that the cellscontained in the implant can bind to the surface Of the biocompatiblesupport or, in certain cases, penetrate into the interior of thissupport.

[0025] This binding of the cells to the support is made possible inparticular by the presence of constituents capable of inducing and/orpromoting the inclusion of the cells within a matrix having theconstitution of a gel.

[0026] This inclusion in the matrix, called gelation, permits theorganization of the cells in a three-dimensional structure in anamorphous environment, not giving rise in vivo to prolongedinflammation.

[0027] According to a first embodiment of the invention, the implantobtained is constituted on the one hand of a biocompatible material suchas a support consisting of a synthetic biocompatible material, inparticular polytetrafluoroethylene fibers (PTFE) or a support consistingof a calcium-based material, in particular a material based on calciumcarbonate, of biological origin, preferably coral and, on the otherhand, a gel optionally loaded with cells expressing the substance ofinterest in particular recombinant cells.

[0028] The invention also relates to a method of preparation of theimplant. The method comprises the steps of:

[0029] placing the biocompatible support in contact with said cells anda constituent capable of inducing and/or promoting their gelation;

[0030] incubation Of the preparation obtained in the preceding step inorder to obtain the gelation of said constituents;

[0031] culture of cells thus obtained under conditions permitting theirbinding to the gelled constituents; and

[0032] recovery of the implant thus obtained.

[0033] a) Implant Consisting of a Synthetic Biocompatible Material.

[0034] The sect of the invention is an implant or neo-organcharacterized in that it comprises cells expressing the substance ofinterest, in combination with polytetrafluoroethylene (PTFE) fibers andcollagen. On being introduced in vivo such an implant is capable ofconstituting a vascularized neo-organ, individualized within a tissue.

[0035] Advantageously, the implant such as described above comprises inaddition a growth factor, for example the bFGF (basic Fibroblast GrowthFactor). This growth factor promotes the vascularization of theneo-organ when the latter is implanted in vivo.

[0036] Preferably, the neo-organ is introduced into the peritonealcavity of the patent in whom it is desired to obtain the expression ofthe exogenous nucleotide sequence contained in the vector of theinvention. This localization of the neo-organ is favourable for thedevelopment of its vascularization and to its maintenance in anindividualized form in this organ. The implantation of the neo organcomprising recombinant cells according to the invention may be permanentor, on the contrary, temporary, it being possible in this latter case towithdraw the neo-organ after a given time of implantation.

[0037] Furthermore, the invention relates to a process for thepreparation at an implant defined above, comprising the steps of

[0038] placing recombinant cells in contact with a solution of collagenand PTFE fibers preferably previously treated with collagen andoptionally with an angiogenic growth factor,

[0039] incubation of the preparation obtained in the preceding step inorder to obtain the gelation of the collagen,

[0040] culture of the cells thus obtained for a time sufficient to allowthe cells to bind to the collagen fibers,

[0041] recovery of the implant thus obtained.

[0042] b) Implant Consisting of a Biocompatible Material of BiologicalOrigin.

[0043] Preferably, the material used is a high porosity coral, i.e. itpossesses a porosity such as that described in the French patent No. 2460 657. It may also be a powder of sufficient porosity to make itpossible to obtain a solid framework for the purpose of constituting acellular network comprising or not a biological support such ascollagen. In the case in which the coral is replaced by collagen,cross-linked or not, this latter may also serve both as support and asmatrix promoting the biological gelation for the constitution of thenetwork of cells.

[0044] According to an advantageous embodiment of the invention the highporosity coral used is of the spherical type.

[0045] According to another embodiment, the implant consists of asupport of biological origin forming a solid matrix, said supportcontaining:

[0046] collagen with variable cross-linking for example in the form offibers or sponges such as those sold under the trade marks Hemostagene®or Paroguide®;

[0047] powdered or fragmented bone; or

[0048] carbohydrate-based polymers such as dextran or hyaluronic acid.

[0049] According to an advantageous embodiment of the invention, thesolid framework of biological origin is capable of being resorbed invivo.

[0050] The implant thus constituted allows the in vivo formation of anindividualized and, if necessary, stable neo-organ in which thebiological support is capable of being progressively resorbed leaving inplace a structure containing a vascularized connective tissue of a formquite comparable to that of the implant, this neo-organ having however,optionally a reduced volume compared with the volume of the originalimplant.

[0051] A particularly preferred implant in the framework of theinvention is such that the constituents capable of inducing and/orpromoting the gelation of the cells are for example uncrosslinkedcollagen or alginates.

[0052] In particular, recourse will be had to type I collagen, forexample, in particular at a concentration of 1.5 mg/ml.

[0053] Other constituents for gelling the cells may naturally be usedprovided that they have the desired functional property and that theyexhibit the properties of biocompatibility required in order to beintroduced in vivo in man or animals.

[0054] c) Use of the Implant of the Invention.

[0055] An implant particularly suited to the realization of theinvention is additionally characterized in that the cells arerecombinant cells carrying genetic information foreign to their genomeand capable of expressing this additional information in vivo and havingthe capacity to be tolerated immunologically by an organism to whichthey might be administered.

[0056] In a preferred manner, an implant according to the invention ischaracterized in that the recombinant cells are modified by a vectorcontaining one or more exogenous nucleotide sequences, coding for anantigen or antigenic determinant or coding for a polypeptide orglycoprotein, soluble in serum, for sample a polypeptide or aglycoprotein of therapeutic interest, in particular a hormone, astructural protein or glycoprotein or a metabolic protein orglycoprotein or a viral protein or glycoprotein or a protein having theproperties of an antibody or antibody fragment.

[0057] In a preferred manner, the implant contains in addition one ormore angiogenic factors, in particular bFGF, preferably incorporatedduring the placing of the biocompatible support in contact with thecells and the constituent capable of inducing and/or promoting theirgelation.

[0058] The binding at the angiogenic factor, for example bFGF, ispromoted by the presence of the gelling agent.

[0059] This agent contributes to the stable binding of the angiogenicfactors to the support of biological origin of the implant.

[0060] In a particularly advantageous manner, the implant also containsheparin or a heparin derivative such as heparan sulfate or heparinfractions.

[0061] Heparin and its derivatives are capable of binding to theconstituents promoting gelation and thus of increasing the affinity ofthe angiogenic factors for the constituents responsible for gelation. Inparticular, heparin binds to collagen and thus increases the affinity ofbFGF for collagen.

[0062] The implant awarding to the invention may be employed in apermanent or temporary fashion for use in man or animals. In fact, itscapacity to constitute in vivo an individualized neo-organ makes itpossible to withdraw it if necessary.

[0063] In a particularly valuable manner an implant according to theinvention can be used for:

[0064] the treatment of genetic diseases, in particular for thetreatment of lysosomal overload, hemophilia A or hemophilia A ofbeta-thalassemia, the exogenous nucleotide sequence contained in therecombinant cells corresponding respectively to those which code forbeta-glucuronidase, for the factor VIII factor IX or erythropoietin, orfor an active part of these sequences;

[0065] the treatment of acquired diseases, for example for the treatmentof viral diseases, in particular for the treatment of an infection dueto the HIV retrovirus for example by the expression and secretion intothe serum of soluble CD4 molecules or of a soluble anti-viral protein;

[0066] the treatment of tumors, the exogenous nucleotide sequencecontained in the recombinant cells coding for a substance capable ofpromoting or enhancing the immune response against the tumor cells.

[0067] The object of the invention is also a composition characterizedin that it contains an implant according to the invention and one ormore other constituents such as for example an antigen, an adjuvant inparticular for vaccination.

[0068] The agents described in the invention thus make it possible toenvisage the treatment of genetic or acquired diseases over a longperiod, more than several months, and do so without the repeatedadministration of the expression product of the exogenous nucleotidesequence whose therapeutic activity is desired Furthermore, the agentsdescribed in the invention lead to the production in the form of asecreted protein of a quantity of expression product of the exogenousnucleotide sequence sufficient to have a therapeutic effect in vivo.

[0069] Another application of the recombinant cells acceding to theinvention or compositions or implants containing them relates to thepreparation of antibodies against the expression product of theexogenous nucleotide sequence contained in the vector introduced intothe recombinant cells. Preferably, these antibodies are produced in vivowhen the recombinant cells are implanted in an organism.

[0070] This use makes it possible for example to produce antibodiesagainst an antigen neither whose amino acid sequence nor necessarily theDNA or cDNA sequence has been identified. Depending on the effectdesired, the introduction of the recombinant vector according to theinvention will be made into a defined cell type.

[0071] Similarly, the invention permits the in vivo production ofantibodies against a defined antigen for example for the purposes ofvaccinating a patient.

[0072] Recombinant cells such as those described below or compositionsor implants containing them may also be used for the treatment oftumors, the exogenous nucleotide sequence contained in the recombinantcells coding far a substance capable of promoting or enhancing theimmune response against the tumor cells.

[0073] As a particular instance, the recombinant cells utilizable forthe treatment of tumors may be cells obtained by recombination of tumorcells taker from a patient with a retroviral vector complying with thespecifications given in the following pages.

Kit According to the Invention

[0074] The invention also includes a kit for the preparation of animplant for achieving in vivo the expression and secretion by cells of asubstance for producing the desired therapeutic effect. The kit of theinvention usually contains:

[0075] a biocompatible support making possible the anchoring of saidcells; and

[0076] a constituent capable of inducing and/or promoting the gelationof said cells.

[0077] In particular, the biocompatible support comprises at least oneof the ingredients chosen from among the group including PTFE or asupport of biological origin, particularly a calcium-based, and inparticular a calcium carbonate-based, support of biological origin, andpreferably coral. The constituent capable of inducing and/or promotingthe gelation of the cells is preferably collagen, in particular type Icollagen, and preferably at a concentration of the order of 1.5 mg/ml.

[0078] The kit according to the invention may also include a DNAcomprising a sequence coding far the substance expressed and secreted bysaid cells. This DNA may be used to transform the cells taken from thepatient to be treated.

[0079] More particularly, the DNA farming part of the kit is aretroviral vector such as that described below.

[0080] The kit may also contain cells having the capacity to express andsecrete naturally or after recombination a defined substance, farexample a substance of therapeutic interest. this deletion makes itpossible to diminish and preferably to neutralize the transcriptionaleffect of the LTR region by conserving the capacity of the exogenouspromoter to promote and control the expression of the exogenousnucleotide sequence contained in the retroviral vector.

[0081] It is also possible to envisage the deletion of all of thesequence constituting the promoter and the enhancer. However, such adeletion has the disadvantage of being accompanied by a considerablediminution of the vector titers.

[0082] According to a variant of the embodiment of the invention, theproviral sequence upstream from the exogenous promoter is the nucleotidesequence situated between nucleotides 1 and about 1500 according to thenumbing of the sequence shown in FIG. 1. This sequence upstream from thepromoter essentially lacks the entire complement of gag, pol and envgenes of the proviral sequence.

[0083] The erogenous nucleotide sequence is advantageously insertedunder the control of the exogenous promoter in the place of the deletedgag, pol and env sequences.

[0084] When the promoter is the PGK-1 promoter described below, theexogenous nucleotide sequence is advantageously inserted at the BamHIsite downstream from this promoter.

2) Exogenous Nucleotide Sequence

[0085] An “exogenous nucleotide sequence” according to the invention isa sequence which is not expressed naturally in a cell in which theretroviral vector of the invention is introduced or is expressed therein insufficient quantity or is one which it is desired to produce ingreater quantity than is normally expressed. This defective orinsufficient expression results from the nature of the cell or occursbecause of a disease affecting this cell in a given individual.

[0086] In a preferred manner, the “exogenous nucleotide sequence” codesfor a defined polypeptide. By “polypeptide” is meant any amino acidsequence irrespective of its size, this expression comprising proteinsand peptides. The polypeptide may be in a glycosylated ornon-glycosylated form

Retroviral Vector and Recombinant Cells

[0087] a) Retroviral Vector

[0088] A novel recombinant retroviral vector according to the inventionis characterized in that it comprises:

[0089] a provirus. DNA sequence modified in that:

[0090] the gag, pol and env genes have been deleted at least in part inorder to obtain a proviral DNA incapable of self-replication, this DNAin addition not being able to recombine to form a wild-type virus;

[0091] the LTR sequence bears a deletion in the U3 sequence such thattranscription of the mRNA it controls is significantly reduced, by atleast 10 fold for example, and

[0092] this recombinant retroviral vector comprising in addition anexogenous nucleotide sequence under the control of a promoter forexample an erogenous inducible or constitutive promoter.

[0093] The principal elements forming this vector are described below indetail.

[0094] 1) Proviral DNA

[0095] A “proviral DNA sequence” is a sequence of DNA transcribed fromthe genomic RNA of the virus when it is integrated in the host cells ofthe virus The proviral DNA thus comprises sequences coding for the gag,pol and env proteins of the retrovirus which correspond respectively tothe nucleoproteins, polymerases, envelope proteins and glycoproteins.

[0096] The retroviral vector according to the invention is such that thegag, pol and env genes of the proviral DNA have been deleted at least inpart in order to obtain a proviral DNA incapable of self-replication,this DNA in addition being unable to recombine to form a wild-typevirus.

[0097] The proviral DNA also bears sequences called LTR (Long TerminalRepeat) which bear regions called R, U3 and U5. These sequences of theLTR region are involved in the replication cycle of the retrovirus.

[0098] The LTR sequence of the proviral DNA has also been mutated bydeletion, for example in its U3 part; this deletion affects thefunctions of the internal enhancer of the LTR region. Furthermore,

[0099] 3) Exogenous Promoter

[0100] The promoter used for the construction of recombinantretroviruses according to the invention is advantageously exogenous withrespect to the sequence whose expression it controls in the sense thatit is not naturally associated with it. It is also possible to use thepromoter of the transferred exogenous gene

[0101] The promoter used in the vector of the invention may be of theinducible or constitutive type This exogenous promoter controls theexpression of the exogenous nucleotide sequence The promoter mayoptionally be accompanied by a regulatory nucleic acid sequence forexample an “enhancer” which would serve to regulate its activity.

[0102] In the case of inducible promoters, promoters such as the Mxpromoter in mice, the promoters including a tetracycline operator oreven promoters regulated by hormones, in particular steroid hormones,may also be used. As regards the so-called “constitutive” promoters, theuse of active internal promoters in resting fibroblasts such as the PGKpromoter or another promoter of the housekeeping gene is preferred.

[0103] In a particularly advantageous manner for the construction of theretroviral vector according to the invention, the exogenous constitutivepromoter is a promoter without a TATA box and in particular the promoterof the phosphoglycerate kinase gene (PGK-1). This promoter is either themouse promoter or the human promoter as described by Adra et al. (Gene60, 1987, 65-74).

[0104] The insertion of the PGK-1 promoter into a retroviral vectormakes possible the transfer of the gene into skin fibroblasts forexample and the stable expression at high level and for the very longterm of this gene after the fibroblasts have been implanted in vivo in arecipient, a mammal for example

[0105] Other constitutive promoters may be used in place of the PGK-1promoter. In general, recourse will be had to a promoter active in thecells which it is desired to transform with the retroviral vector and inparticular active promoters in quiescent fibroblasts, for examplepromoters preceding the genes for the cytoskeleton. It is also possibleto mention the promoter of beta-actin. (Kort et al., 1983, Nucl. AcidsRes. 11: 8287-8301) or the promoter of the vimentin gene (Rettlez etBasenga (1987), Mol. Cell. Bid. 7: 1676-1685). The promoter used maylack a “TATA box”.

[0106] 4) Preferential Comstructions of the retroviral Vector

[0107] A first particular retroviral vector in the framework of theinvention is such that an exogenous nucleotide sequence and an exogenousconstitutive promoter as well as the proviral DNA sequence are borne bya plasmid. As an example, these sequences may be borne by the plasmidpBR322 which allows the introduction of the DNA into the cell lines inwhich it is desired to produce the vector.

[0108] According to another advantageous embodiment of the invention avector complying with the preceding specifications is in additioncharacterized in that the proviral DNA is derived from the Mo-MuLVretrovirus. Other retroviruses of the MuLV family may be used andmention should be made, for example, of the HaSV or F-MuLV retroviruses.

[0109] Preferably, the sequences of the pol and env genes of theproviral DNA are completely deleted. In this case, the sequence of thegag gene may also be entirely deleted or, on the other hand, beconserved in part, provided that the proviral DNA thus constituted is nolonger capable of replication.

[0110] Advantageously, a retroviral vector such as previously defined ischaracterized in that the U3 region of the LTR3′ fragment is deleted atnucleotide 2797 of FIG. 1. This deletion corresponds to a deletion of afragment situated between nucleotides 7935 and 8113 according to thenumbering of the sequence published by Shinnick et al. (Nature 293,543-548, 1981). This deleted fragment of the U3 sequence of the LTR3′contains the enhancer region of the LTR.

[0111] Preferably, a retroviral vector according to the invention is atype pM48 vector derived from the Moloney virus in which the viral“enhancer” localized in V3 has been deleted and which contains aninternal promoter active in resting fibroblasts, preferably the PGKpromoter or another promoter of the housekeeping gene, such as thevector shown in FIG. 2, modified by the exogenous nucleotide sequence atthe BamHI site.

[0112] The retroviral vector M48 LacZ (also designated by the expressionpM48 LacZ was deposited with the CNCM (Collection Nationale de Culturede Microorganismes, Paris-France) under the No. I-1298 an Apr. 16, 1993.

[0113] This vector is derived from the vector pM48 and is characterizedin that it contains downstream from the exogenous constitutive promotera BamHI fragment of the gene for beta-galactosidase.

[0114] This fragment of the beta-galactosidase gene may easily bedeleted and replaced by an exogenous nucleotide sequence of interest,for example a sequence coding for a protein or a glycoprotein capable ofhaving therapeutic value or against the protein product of which itwould be desired for example to obtain antibodies.

[0115] A retroviral vector of the invention may also contain a sequencecapable of enhancing the transcriptional activity of the promoter eitherconstitutively or inducibly. The vector may contain an enhancer sequenceupstream from the exogenous promoter.

[0116] b) Recombinant Cells

[0117] Moreover, the invention relates to recombinant cellscharacterized in that they are cells having the capacity to be toleratedimmunologically by an organism in which they might be implanted,modified by a retroviral vector complying with one of the precedingspecifications

[0118] Such cells may be cells derived from the organism into which theyare to be implanted after recombination by transduction with the vectorof the invention or cells lacking at their surface antigens recognizedby the immune system of the organism into which they are implanted.

[0119] They may also be endothelial cells, myoblasts, muscle cells ortumor cells irradiated or treated according to other procedures toprevent their proliferation and taken from the patient whose geneticmake-up it is desired to modify so that they may counteract thedevelopment of the tumor.

[0120] Preferably, these recombinant cells are recombinant fibroblastsand in particular skin fibroblasts. Preferably, they are autologousfibroblasts with respect to the patient in whom it is desired to implantthem after their modification by a vector according to the invention.Fibroblasts prepared from other organisms may be used such as forexample fibroblasts isolated from an umbilical cord. The geneticallymodified fibroblasts used in the context of the present invention arenot immortalized fibroblasts.

[0121] These cells may be modified by a recombinant vector coding for adefined polypeptide, said vector permitting the expression of theforeign DNA in the cells. They may also be modified by the methods ofso-called homalogous recombination. The penetration of the vector intothe cells is carried out by using electroporation or precipitation withcalcium phosphate or also by any other method implicated in the entry ofa nucleic acid either alone or through the intermediary of a recombinantvirus, for example

[0122] By “recombined cells” is also meant tumor autologous cellsirradiated prior to their introduction into the gel, having the capacityto maintain at their surface tumor antigens accessible to the immunesystem of the host into which the implant according to the invention hasbeen introduced.

[0123] The recombinant cells of the invention are also obtained eitherby infection of the cells to be modified with a retroviral vector of theinvention, or by other transduction methods involving naked DNADNA-proteins complex or an adenoviral vector. In the case of aninfection with a retroviral vector, the sequence coding for thepolypeptide whose expression (exogenous nucleotide sequence) is desiredis introduced into the proviral DNA.

[0124] In a preferred manner, the recombinant cells are modified by avector containing one or more exogenous nucleotide sequence(s) codingfor an antigen or an antigenic determinant or coding for a polypeptideor a glycoprotein, soluble in the serum, for example a polypeptide or aglycoprotein of therapeutic interest, in particular a hormone, astructural protein or glycoprotein or a metabolic protein orglycoprotein or a viral protein or glycoprotein or a protein having theproperties of an antibody or an antibody fragment.

[0125] As an example, this exogenous nucleotide sequence codes forbeta-glucuronidase or another lysosomal enzyme such as alpha-iduronidaseor arylsulfatase B, a coagulation factor such as factor VIII or factorIX erythrapoietin or any active part of one of these proteins.

[0126] Other advantages and characteristics of the invention will becomeapparent in the Examples and the Figures which follow.

[0127]FIG. 1. nucleotide sequence of the vector M48;

[0128]FIG. 2 representation of the vector pM48.

EXAMPLES Preparation of the Recombinant Vector

[0129] The transfer and expression of exogenous genetic material makesit possible to modify experimentally the properties of the tissue. Ithas been possible to suggest treatments based on this principle fordiseases due to genetic deficiencies or acquired diseases. The exampledescribed below makes use of a retroviral vector for introducing intocells the genetic information coding for a secreted protein. Thereimplantation in the organism of the genetically modified cells iscarried out by combining these cells with collagen, an angiogenic factorand the framework of the coral or cross-linked collagen type Thelong-term in vivo expression of the genetic material thus transferred isobtained by using the promoter of the murine phosphoglycerate kinasegene.

[0130] 1—Retroviral Vector

[0131] 1.1.—Properties

[0132] The retroviral vector M48 (FIG. 2) is instructed from proviralsequences is dated from the genomic DNA of rat cells infected with theMoloney murine leukemogenic retrovirus (Mo-MuLV). The gag, pol and envgenes which code for the proteins of the virus were deleted and replacedby the sequences to be transmitted. The retroviral sequences in cisnecessary for the production and maturation of the transcripts of therecombinant viral genome, for their packaging in infectious viralparticles, their reverse transcription and their integration in thegenome of the infected cell have been conserved The sequences of therecombinant provirus and the flanking fragments at cellular DNA areborne by the bacterial plasmid pBR322.

[0133] The promoter of the mouse phosphoglycerate kinase (PGK-1) genewas inserted in the place of the viral genes A unique BamHI cloning siteenables the sequences to be transmitted to be placed in the retroviralvector under the control of this promoter.

[0134] This retroviral vector bears a deletion in the U3 sequences ofthe internal LTR (Cone et al., Mol. Cell Biol. 7, 887-897, 1987) Thisleads to a considerable reduction of the mRNAs being initiated from the5′ LTR and to a predominance of mRNAs produced under the control of thePGK-1 promoter. The use of this vector hence results in the insertion inthe genome of the target cell of an expression cassette of the sequenceof interest under the control of the PGK-1 promoter.

[0135] 1.2.—Structure of the Vector

[0136] The complete sequence of the vector M48 (FIG. 1) comprises

[0137] Position 1 to 2019: Mo-MuLV sequences comprising the 5′ longterminal repeat (or 5′ LTR or LTR 5′), the packaging sequences ψ+ whichinclude a part of the gag gene (position −447 to 1564 in the numberingof Shinnick et al. (Nature 293, 543-548, 1981). These sequences aremodified as follows: insertion of a SacII linker (5′ CCCGCGGG3′) atposition 1074 (position 626, Shinnick et al.).

[0138] This leads to a mutation in phase with the sequence coding forgag.

[0139] Position 2020 2526: Promoter of the mouse gene coding forphosphoglycerate kinase (position −524 to −20, before the translationstart codon, according to Adra et al., Gene 60, 65-74, 1987).

[0140] Position 2527 to 2532: Unique BamHI site for the insertion ofsequences which will be expressed under the control of the PGK-1promoter.

[0141] Position 2533 to 3101: Mo-MuLV sequences including the 3′ end ofthe env gene (32 codons) and the LTR3′. These sequences are modified asfollows: deletion of the U3 sequences of the LTR included between 7935and 8113 in the numbering of Shinnick et al. and the insertion of alinker SalI (2798 in the present numbering).

[0142] Position 31012 to 3674: Rat genomic sequences.

[0143] Position 3675 to 8003: Sequences of the plasmid vector pBR322(position 4363 to 29) comprising the beta-lactamase gene These sequencesare modified as follows: destruction of the BamHI site (position 7657,present numbering).

[0144] Position 8004 to 8388: Rat genomic sequences.

[0145] 1.3.—Production of the Retroviral Vector

[0146] This production is carried out by introducing the recombinantproviral structure in a cell line in which the gag, pol and env genesare expressed constitutively.

[0147] This line, called a transcomplementary or packaging line,synthesizes retroviral particles lacking genomic RNA The ψCRIP linederived from NIH/3T3 mouse fibroblasts is used here (Danos and Mulligan,Proc Natl. Acad. Sci. (USA) 85, 6460-6465, 1988). When the recombinantconstruction is introduced by transfection, the RNA transcripts from theLTR5′ are encapsidated and the cells then produce particles capable oftransmitting the recombinant genome but which are incapable ofreplicating.

[0148] After transfection of the recombinant construction the cellsproducing retroviral vector are selected Either a clone producing highviral titers or a polyclonal population is used for the preparation ofthe vector. The steps of isolation of a producing clone are thefollowing:

[0149] 1) The ψCRIP cells are transfected by a coprecipitate withcalcium phosphate (Graham and van der Erb, Virology, 52, 456, 1973) ofthe retroviral construction and the plasmid pSV2neo (Southern and Berg,J. Mol. Appl. Gen. 1, 327-341, 1982) in a molar ratio of 10 to 1.

[0150] 2) The cells which have integrated in a stable manner and expressthe exogenous DNA are selected in the presence of 1 mg/ml G418(Geneticin, Gibco).

[0151] 3) The population of cells resistant to G418 in which isolatedindividual clones are tested for ther capacity to produce the retroviralvector. For that purpose, NIH/3T3 target cells are placed in thepresence of culture medium recoated from the ψCRIP cells, and thetransmission of the recombinant provirus is analyzed after 48 hours. Theanalysis, performed on the genomic DNA of the target cells, is made byPCR (qualitative method) or by Southern blot (quantitative method).

[0152] 4) A library of producing cells is stored at −135° C.

[0153] 2—Preparation of the Stocks of Retroviral Vector

[0154] The culture supernatant of the packaging cells is recoated afteran incubation for 24 hours with the packaging cells producing theretroviral vector and placed in contact with the target cells in orderto achieve the infection.

[0155] 2.1.—Bulk Preparation

[0156] A stock of a hundred ampoules containing producing cells isstored at −135° C. After being thawed, the cells are amplified for 10days, successively in bottles then in roller bottles of 875 cm², until20 confluent rollers are obtained. The production is then begun. Itlasts 4 or 5 days. The culture medium is DMEM containing 1 g/l ofglucose, 10 mM of sodium pyruvate and supplemented with 5% newborn calfserum (Hyclone). Each roller bottle makes possible the conditioning of100 ml of medium per 24 hours. This culture supernatant is centrifugedto remove the cell debris. The weekly production is 8 to 10 liters ofviral supernatant.

[0157] 2.2.—Concentration

[0158] The supernatant may be concentrated to increase the infectioustiter of the retroviral vector. After centrifugation, the supernatant isdialyzed tangentially against a Sartocon membrane of porosity 100.000 byusing the Crossflow apparatus of Sartorius. A twenty fold concentrationis obtained in 3 hours. It is accompanied by a 20 increase in theinfectious titer. After concentration, the viral preparation is usedimmediately or stored at −80° C. if it is not to be used for at least 2weeks.

[0159] 3—Skin Fibroblasts

[0160] 3.1.—Isolation and Placing in Culture

[0161] The fibroblasts intended for the gene transfer are obtained by acutaneous biopsy performed under very careful aseptic conditions. In themouse, a syngeneic animal is sacrificed for this purpose. In the largemammal, one or more fragments of skin about 10 cm² are taken undergeneral anesthesia. After sectioning in strips of several mm², thefragments are dissociated by an enzymatic treatment. For a fragment of10 cm², the reaction is carried out for 2 hours at 37° C. by gentleshaking in 50 ml of RPMI 1640 medium supplemented with 10% fetal calfserum, 100 mg of collagenase (Worthington), 500 U of dispase(Collaborative Research Inc.). After centrifugation and washing thecells are counted then seeded in 150 cm² bottles at a concentration of 1to 5×10⁶/cm². The culture medium is RPMI 1640 supplemented with 10 to20% fetal calf serum.

[0162] 3.2.—Infection by the Retroviral Vector

[0163] The infections with the preparation of retroviral vector arestarted the day after the placing in culture although the cell densityis less than 20% of confluence They are repeated daily following thesame protocol for 4 or 5 days. After removal of the culture medium, thecells are placed in contact with the concentrated or unconcentratedretroviral vector preparation, bit supplemented in all cases with 8μg/ml of polybrene The incubation is carried out for 2 hours at 37° C.,then without prior washing the viral preparation is replaced by theculture medium.

[0164] When a vector coding for a protein detectable under themicroscope is used, it is observed that this process allows theintroduction of the foreign gene into more than 90% of the fibroblasts.The level of secretion of the protein into the culture supernatant maybe checked during and at the end of this process All or part of thecells may be frozen at this stage.

[0165] After infection, the cells are amplified to produce a numbersufficient far reimplantation, i.e. 2-6×10⁸ cells/kg of body weight. Theamplification is carried out on culture trays (multitray, Nunc).

[0166] 3.3.—Harvesting of the Genetically Modified Cells

[0167] After amplification, the cells are harvested by treatment withtrypsin. After being washed and counted, they are available to be usedfor the formation of a neo-organ. A sample is stored to check thegenetic transfer by Southern blot as well as the expression andsecretion of the foreign protein by a suitable procedure

Example 1

[0168] Secretion of a lysosomal enzyme (beta-glucuronidase) in the mousewith implants containing PTFE fibers, rat tail collagen and skinfibroblasts modified with a retroviral vector.

[0169] 1.1.—Constituents

[0170] Three constituents are assembled in vitro. The method describedbelow is applicable to the construction of neo-organs of 1 ml containing5×10⁶ to 10⁷ genetically modified cells. The same proportions are usedfor the formation of neo-organs of large size containing 1 to 5×10⁹cells.

[0171] 1) Polytetrafluoroethylene fibers (Gore and associates)sterilized by autoclaving (120° C., 30 minutes) are coated by type 1collagen. The fibers are bathed in a 0.1% solution of rat tail collagen(Sigma) in 0.1 N acetic acid. This treatment is carried out under vacuumfor 1 hour at room temperature in order to expel air present between thesynthetic fibers. The collagens of mouse, bovine or human origin may beused indiscriminately. After drying for 24 hours under a hood withlaminar air flow, the fibers thus surrounded with a fine film ofcollagen are incubated in the presence of an angiogenic growth factor(basic FGF, Farmitalia, 10-20 ng/50 mg of fibers in a solution of PBS5%beta mercaptoethanol). The fibers thus treated are washed in PBS andstored at 4° C.

[0172] 2) A solution constituted of the following ingredients isprepared For 1 ml: 100 μl 10×RPMI 1640 (Gibco); 12 μl 7.5% bicarbonate;8 μl, 7.5 N NaOH; 2.5 1, 1M Hepes; 100 μg penicillin/glutamine (Gibco);10 ng bFGF (Farmitalia); 1.5 mg collagen (Bioethica); distilled water to1 ml. This solution of syrupy consistency is stored at 4° C.

[0173] 3) After washing, the genetically modified cells are resuspendedin 100 μl of RPMI 1640 with 15% fetal calf serum

[0174] 1.2.—Assembly

[0175] The in vitro assembly of the neo-organ is carried out understerile conditions in a hood, in the following manner:

[0176] 1) 1 mg of treated fibers are spread out in a culture dish(Corning 24 wells plate).

[0177] 2) The genetically modified cells (100 μl) are incorporated intothe collagen solution (1 ml) using a shaker,

[0178] 3) This mixture is deposited on the fibers

[0179] The entire preparation is placed in an incubator (37° C., 5% CO₂)for 30 minutes in order to obtain the gelation of the collagen. Whenthis occurs, 1 ml of RFMI 1640 with fetal calf serum is deposited on thegel in order to compensate the variations in pH. Two to four hourslater, the gel is delicately detached from the rim of the culture dishwith the aid of an injection needle and transferred to a larger dish inthe presence of an excess of culture medium for a period of 12 to 24hours. During this lapse of time the fibroblasts included in thecollagen gel bind to the collagen fibers and this induces a retractionof the structure. It is usual to observe a reduction of more than 50% ofthe initial volume The whale is then ready to be implanted in theorganism.

[0180] 1.3—Implantation

[0181] The neo-organ is introduced in the peritoneal cavity in thecourse of a laparotomy performed under general anesthesia. In the mouse,the implantation is effected by inserting, without binding the neo-organbetween the midgut loops in contact with the mesentery. In the largemammal, the neo-organ is fixed by two points between the two layers ofthe omentum. Vascular connections are established from the first daysonwards. The exogenous protein may then be released into the circulationof the recipient animal. As from the third week following theimplantation, an organ often pediculate, encapsulated and wellindividualized is formed. Very many vascular connections are visible atits surface. Inflammatory adhesion does not exist. The examinations at 3and 6 months show an identical appearance. The histological analysis atthis stage reveals the presence of loose, vascularized and only slightlyinflamed connective tissue

[0182] 1.4—Treatment of Diseases of Lysosomal Overload

[0183] This process was applied to the treatment of a disease oflysosomal overload for which a mouse model is available. It involves adeficit of beta-glucuronidase responsible for a mucopolysaccharidosis.The beta-glucuronidase is phosphorylated and is thus in part transportedto the lysosomes and in part secreted in the circulation. It may bepicked up by other tissues through the intermediary of themannose-6-phosphate receptor expressed at the surface of many cell. Thisspecific recovery permits a therapeutic approach in which the continuoussystemic distribution of the missing enzyme is ensured by geneticallymodified cells.

[0184] Implants of 0.5 cm³ containing 20×10⁶ autologous fibroblasts arewell tolerated by the mouse. The implanted cells express the humanbeta-glucuronidase gene inserted into the vector M48 under the controlof the PGK promoter and secrete the foreign protein for more than 5months. The biological effect is expressed by a rapid fall in theurinary elimination at the intermediary catabolites of the degradationof the mucopolysaccharides. The histological normalization of thetissues of the recipient animal is spectacular, particularly in theliver and the spleen. It is stable with time and depends on the presenceof the neo-organ whose excision induces a return to the initialpathological state.

[0185] This process is applicable to the treatment of all of thediseases o lysosomal overload, including Gaucher's disease for which thesupply of a soluable form of the glucocerebrosidase can be envisaged.

Example 2

[0186] Secretary of a lysosomal enzyme (beta-glucuronidase) in the dogwith implants containing PTFE fibers, rat tail collagen and skinfibroblasts modified with a retroviral vector.

[0187] Four dogs received from 1 to 6 neo-organs each containing 10⁹autologous fibroblastsgenetically modified by means of the vectorM48-βglu in order to secrete human beta-glucuronidase. Three animals ofthe Labrador race weighing 21 to 29 kg and one animal of the Beagle raceweighing about 9 kg were used. The surgical implantation performed undergeneral anesthesia consisted of inserting the implants between the twoparietal and visceral layers of the omentum close to the greatercurvature of the stomach. This rapid operation is accompanied by simpleoperative follow-ups and makes it possible to envisage in the future animplantation by coeliosurgery. One and a half months, four months andsix months after the implantation an exploratory laparotomy wasperformed in order to make a macroscopic check-up of the implant. Theidentification of the omentum was easy and the detection of the graftedstructure was made very quickly. No adhesion was recorded and thevascularization was considerable, including large calibre vessels inconnection with the implant. The appearance of the implant has notaltered during the period co this observation. A liver biopsy practicedat the same time made it possible to investigate the presence of humanbeta-glucuronidase. The measurement of the activity on cell extractsshowed a level equivalent to 2-3% of the endogenous canine activitywhich was stable with time in the animals having received more than2×10⁹ genetically modified autologous fibroblasts. The revelation insitu on sections showed an activity localized in the Küpffer cells. Theresults are summarized in Table I. TABLE I Human beta-glucuronidaseabsorbed by the liver of dogs in which neo-organs were implantedActivity of human beta-glucuronidase Number of cells % of endogenouscanine activity In situ revelation on sections Animal Weight implantedday 0 day 45 day 120 day 180 day 0 day 45 day 120 day 180 Nougatine 28kg 0.8 × 10⁹   <0.2 3 0.8 − − +++ +/− − Eglantine 26 kg 2 × 10⁹ <0.2 2 21 − +++ ++ ++ Doris 21 kg 3 × 10⁹ <0.2 3 3 1 − +++ +++ ++ Cyclo 10 kg 6× 10⁹ <0.2 nd 1 nd − nd nd nd

Example 3

[0188] Secretary of a lysosomal enzyme (beta-iduronidase) in the mousewith implants containing PTFE fibers, rat tail collagen and skinfibroblasts modified with a retroviral vector.

[0189] A cDNA coding for the human beta-L-iduronidase was introducedinto the vector M48 and a recombinant retrovirus was produced in theψCRIP line. Fibroblasts of nude mice were placed in primary culture andinfected with this retroviral vector. The cells secreting the humanenzyme were introduced into six syngeneic recipients in two neo-organseach containing 10 millions cells.

[0190] The animals were sacrificed after 35 to 77 days and the presenceof the human enzyme in their liver and spleen was verified by means of amonoclonal antibody. An enzymatic activity equivalent to 1-2% of theendogenous activity was demonstrated, indicating the production of theenzyme from the cells of the neo-organ and its detection at a distance.

Example 4

[0191] Secretary of erythropoietin (EPO) in the mouse with implantscontaining PTFE fibers, rat tail collagen and skin fibroblasts modifiedwith a retroviral vector.

[0192] The nlslacZ gene was excised from the vector M48-nlslacZ by BamHIdigestion and replaced by a cDNA coding for mouse EPO synthesized by PCRthus generating the vector M48EPO. A done d ψCRE cells producing M48EPOretroviral particles was isolated. A sample of fibroblasts was takenfrom adult mice by skin biopsy and a primary culture was established inRPMI 1640 medium with 10% FCS. These cells were infected by the vectorM48EPO repeatedly for the first 4 days of the culture Analysis bySouthern blot showed that the infected cells contained on average 2copies of the M48EPO genome per cell. Analysis by Northern blot showedthe very predominant expression of the cDNA of the EPO under the controlof the PGK-1 promoter. A biological assay of the EPO activity secretedby these cells as well as an ELISA assay measured a secretion of 17units of EPO per million cells per 24 hours. These cells were amplifiedin culture, trypsinized, then resuspended in RPMI 1640 at aconcentration of 2.5×10⁷ to 2×10⁸ cells per ml.

[0193] Implants were prepared 1 combining the following ingredients in a0.9 cm² well: 1) PTFE filers treated previously as described above to becoated successively with rat tail collage (Sigma), heparin (Roche) andbFGF (Promega); 2) 1 ml of a solution containing x mg/ml of rat tailcollagen (Sigma), bFGF (10 μg/ml) in RPMI 1640 medium, 3) a volume of 10μl of the suspension of fibroblasts genetically modified to secrete EPO.After having obtained a homogeneous mixture, this latter is solidifiedby incubation at 37° C. far 30 minutes, then the gel is very carefullyseparated from the walls of the culture well, transferred to a 35 mmdiameter Petri dish coated with RPMI 1640 medium containing 10% FCS andincubated at 37° C. for 3 days. A contraction of the gel occurred duringthis incubation resulting in a 50% diminution in size The implants werethen inserted in the peritoneal cavity of 14 syngeneic mice withgenetically modified fibroblasts.

[0194] The weekly measurement of the hematocrit (normal value 46+/−1.5%)showed a progressive rise towards a plateau, the level of which variedas a function of the number of cells sting EPO in the implants. Thisplateau was 80% for 2×10⁷ cells, 70% for 10⁷ cells, 60% for 5×10⁶ cells,52% for 25×10⁶ cells. It was maintained at this level during the 6months period of observation of the animals. The concentrations of EPOin the serum of the mice bearing implants secreting EPO varied from 60to 400 mU per ml (normal <20 mU/ml).

[0195] The implants constituted of PTFE, rat tail collagen and cellsgenetically modified by the retroviral vector M48EPO thus make possiblea stable long-tm secretion of EPO in vivo at high levels estimated to bebetween 500 and 1500 mU/kg/24 hours, i.e. sufficient in man for thecorrection of the anemia due to hemoglobinopathies such as sickle cellanemia and beta-thalassemia. Levels 10 fold lower would be sufficientfor the treatment of anemia associated with chronic renal insufficiency.

In vivo Utilization of Implants Containing Coral as Biological Support

[0196] 1.In the Mouse

[0197] In the C3H/He mouse compatibility experiments with Biocoral® asimplant showed that it could represent an advantageous alternative tothe synthetic supports.

[0198] It was in fact possible to coat Biocoral® effectively withdifferent ingredients of the extracellular matrix as well as angiogenicfactors. It was shown that murine type I collagen at a concentration of0.5 mg/ml in 0.001% acetic acid adheres and covers the coral supportafter drying in air at room temperature for 12 to 24 hours. The presenceof this element of the extra-cellular matrix then made possible theirreversible binding of the basic fibroblast growth factor labelled with1251 (¹²⁵I-basic Fibroblast Growth Factor) (bFGF) to Biocoral® In thisway the binding of an average of 50 ng of bFGF to 40 mg of coral supportpretreated with murine type I collagen was achieved. Although thisbinding could have also been done in the absence of pretreatment withcollagen, in this latter case it was reversible, hence of low affinity.Collagen is thus an essential intermediary and justified for the stablebinding of angiogenic factors to Biocoral. Finally, it was possible toincrease the binding of bFGF five to ten fold when the coating of 40 mgof Biocoral by collagen was supplemented by 200 μg heparan sulfate(Sigma) (1 mg/mi solution in PBS) or 500 units of heparin (Roche) (5000units/ml solution). These two components of the extracellular matrixhave a known affinity for collagen (Baird, A, Schubert, D., Ling, N. andGuillemin, R (1988) Receptor and heparin binding domains of basicfibroblast growth factor. Proc. Natl. Acad. Sci. USA 8 2324-2328).

[0199] Their anionic and polysulfated properties are responsible for thestrong affinity for the bFGF (Johnson, D. E., Lee, P. L., Lu, J., andWilliams, L. T. (1990) Diverse fonrs of a receptor for acidic and basicfibroblast growth factors Mol. Cell. Biol. 14 4728-4739). Afterincubation in the presence of one or other of these components I 1 h at4° C. with shaking, the Biocoral was dried in air at room temperatureThus heparinized, the coral has an anti-coagulant action.

[0200] Assays of subcutaneous implantation with 40 mg of high porosity(50%) spherical coral coated with murine type I collagen and bFGF (=50ng/40 mg of coral) were performed in vivo on ten mice. The macroscopicand histological appearance of these implants was analyzed over a periodof 7 months. The results show that Biocoral was progressively resorbedduring several months and was replaced b a structure formed ofvascularized connective tissue having the initial spherical form of thecoral implant with, however, a reduction in volume of approximately athird.

[0201] The histological analysis of this connective tissue at threemonths shaved an intense vascularization and the total absence ofinflammatory cells in spite of the coral residues present. The actualconnective tissue was farmed of only slightly dense mesenchymal cells,surrounded by newly formed collagen fibers. The analysis at 7 monthsshowed similar results without involution.

[0202] 2. In the Dog

[0203] In the 25 kg dog we carried cut a first implantation experimentwith a neo-organ, the external part of which made of porous coral (50%)made it possible to retain a retracted collagen gel containing 1 to2×10⁹ genetically modified autologous fibroblasts. The Biocoral supporthad been coated with murine type I collagen and heparin (Roche). Afterdrying, the coral thus pretreated was incubated in the presence of anangiogenic growth factor of the bFGF type (Farmitalia) then rinsed withPBS. The surgical implantation performed under general anesthesiaconsisted of inserting the whole implant between the two parietal andvisceral layers of the omentum close to the greater curvature of thestomach. This rapid operation made possible simple operative follow-upsand makes it possible to envisage in the future an implantation bycoeliosurgery. Forty five days after, an exploratory laparotomy wasperformed in order to make a macroscopic check-up of the implant. Theidentification of the omentum was easy, no adhesion was noted and thedetection of the coral implant, containing the genetically modifiedautologous fibroblasts, was made very quickly. It was not possible toobserve any adhesion and the vascularization was considerable, includinglarge calibre vessels in connection with the implant. A process ofprogressive resorption of the coral was observed for at least 4 months.

[0204] 3. Other Mammals.

[0205] The creation of neo-organs by using Biocoral suited to surgicalimplantation in large manuals, in particular man, with a view to theirclinical development is carried out analogously. Biocoral can bemodulated in its design, is capable of triggering a sustainedangiogenesis combined with its own resorption and, finally, is devoid ofinflammatory local and systemic effects. Such a biomaterialadvantageously serves as receptacle and skeleton for a type I collagengel containing genetically modified autologous cells, capable ofsecreting a therapeutic factor in the circulation once the vascularconnections are established.

Example 5

[0206] Secretary of erythropoietin (EPO) in the mouse with implantscontaining fragments of coral, rat tail collagen and skin fibroblastsmodified with a retroviral vector.

[0207] In the same manner as in Example 4, mouse fibroblasts wereobtained from a skin biopsy, placed in culture and transduced with thevector M48EPO. After amplification, the cells were trypsinized andresuspended in RPMI 1640 medium containing 10% FCS at a concentration of10⁸ cells per ml.

[0208] Implants were prepared by combining the following ingredients ina 0.9 any well: 1) coral powder (Inoteb) composed of particles ofporosity >45% and a granulometry between 600 and 1000 μm, the pares ofwhich had a mean diameter of 150 μm, previously treated by incubation ina solution of rat tail collagen (1 mg/ml) for 30 minutes at roomtemperature, dried, then incubated in a heparin solution (Roche) for 30minutes at room temperature, dried, then incubated in a 100 μg/mlsolution of bFGF (Promega) for 30 minutes at room temperature, 2) 1 mlof a solution containing 3 mg/ml of rat tail collagen (Sigma), bFGF (100μg/ml) in RPMI 1640 medium; 3) a volume at 100 μl of the suspension offibroblasts genetically modified to secrete EPO. As in the examples 1 to3, a homogeneous mixture of these constituents was solidified byincubation at 37° C. for 30 minutes, then the gel was very carefullyseparated from the walls of the culture wells, transferred to a 35 mmdiameter Petri dish, coated with RPMI 1640 medium containing 10% FCS andincubated at 37° C. for 3 days. The implants were then inserted in theperitoneal cavity of 3 syngeneic mice with genetically modifiedfibroblasts.

[0209] As in Example 4, the measurement of the hematocrit of the animalrecipients showed a rapid increase reaching a plateau after 3 weeks, andwhich was maintained at this level for all of the period of theobservation (2 months). Implants formed of a structured collagen gelwith fragments of coral thus make possible the maintenance In thefunctional state of genetically modified fibroblasts implanted in theperitoneal cavity of recipient animals, as well as the long-termsecretion f a soluble protein in the serum.

Example 6

[0210] Secretary of erythropoietin (EPO) in the mouse with implantscontaining cross-linked collagen fibers, rat tail collagen and skinfibroblasts modified with a retroviral vector.

[0211] A similar experiment to the Examples 4 and 5 consisted ofproducing implants combining mouse fibroblasts transduced with theM48EPO vector, rat tail collagen and a support farmed of cross-linkedcollagen fibers (Imedex). The preparation was made in the followingmanner: 1) the fibers dehydrated and sterilized by radiation were firstrehydrated by incubation for 12 hours at 4° C. in RPMI 1640 medium. Theywere then incubated at roam temperature for 30 minutes in RFMI 1640containing 10% FCS and 100 μg/ml of bFGF (Promega), then loaded into a0.4 cm² well to which was added 1 ml of RPMI 1640 solution containingFCS (10%), rat tail collagen (1 mg/ml), bFGF (100 μg/ml, Promega) and10⁷ fibroblasts genetically modified to secret EPO. After solidificationat 37° C. far 30 minutes, the gel was then very carefully detached fromthe walls of the culture well, transferred to a 35 mm diameter Petridish and coated with RPM 1640 medium containing 10% FCS The implantswere incubated for 3 days at 37° C., moderate contraction of thestructure was observed, then they were implanted in 3 syngeneic mice.

[0212] In a manner similar to that described in the Examples 4 and 5,the weekly measurement of the hematocrit showed a progressive rise untila plateau was reached which was maintained for the duration of theobservation period (2 months). Implants formed of a structured collagengel with cross-linked collagen fibers thus make possible the maintenanceof the functional state of genetically modified fibroblasts implanted inthe peritoneal cavity of animal recipients as well as the long-termsecretion of a soluble protein in the serum

Example 7

[0213] Detoxification of bilirubin by glucurono-conjugation in ratsbearing implants containing PTFE fibers, rat tail collagen andgenetically modified fibroblasts expressing the enzyme UDP-glucuronosyltransferase. Bilirubin is a degradation product of the haem producedcontinuously and in considerable quantity by the organism.

[0214] Bilirubin is liposoluble and can penetrate passively into cellswhere its accumulation is toxic The elimination of bilirubin is achievedby glucurono-conjugation in the hepatocyes by the enzyme bilirubinUDP-glucuronyl transferase (bil. UDPGT) which exists in two knownisoforms (1 and 2). The addition if glucuronic add makes the moleculewater soluble, non-toxic and makes possible its excretion in the bileComplete hereditary deficit of bil. UDPGT activity is responsible for avery serious disease (Crigler-Najjar disease) which requires livertransplantation. There exists a model for this disease in the rat (Gunnrat).

[0215] The rat cDNA coding for the isoform 2 (bil. UDPGT-2) and humancDNA coding for isoform 1 (bil. UDPGT-1) were inserted into the M48vector after excision of the nlslacZ gene by the BamHI enzyme, thusgenerating the vectors M48GT-2 and M48GT-1. In a manner similar to thatdescribed in Examples 1, 3 and 4 samples of Gunn rat fibroblasts weretaken by skin biopsy, placed in culture and transduced by the vectorM48GT-2. The presence of, on average, one copy of the vector genome inthe transduced cells was verified by Southern blot, and the expressionunder the control of the PGK promoter was verified by Northern blot. Thebil. UDPGT enzymatic activity was detected in the microsomal extracts ofthe infected fibroblasts. The cells were amplified in culture,trypsinized then resuspended in RFMI 1640 at a concentration of 2×10⁸cells per ml.

[0216] Implants containing 2×10⁷ genetically modified cells wereprepared in the same manner as in Examples 1, 3 and 4. Two implants wereinserted in the peritoneal cavity of each recipient Gunn rat. As thecolony of Gunn rats used is not inbred, care was taken that each animalis reimplanted with its own cells. The measurement of the unconjugatedbilirubin concentrations in the treated animals and in control rats ofthe same age showed a significant and stable diminution (between 20 and50%) during a period of observation of 2 months in rats bring implantsof fibroblasts expressing the bil. UDPGT. Bile samples were collectedwhen the animals were sacrificed and analyzed by high pressure liquidchromatography (HPLC). Whereas the untreated animals show a totalabsence of conjugated bilirubin in the bile, the Gunn rats bearingimplants containing fibroblasts expressing the bil. UDPGT showed peaksidentified as mono and di-conjugated bilirubin. The proportion ofconjugated bilirubin attained up to 11% of the bilirubin present in thebile.

[0217] These results indicate that rat fibroblasts transduced with thevector M48UDPGT-1 and grafted into Gunn rats in the form of implantscombining PTFE fibers and rat tail collagen are capable of achieving invivo the glucurono-conjugation reaction of bilirubin, and the conjugatedbilirubin can then be eliminated in the bile by the hepatocyes, thusmaking possible a partial correction of the phenotype of the diseasedanimals. It is thus possible to achieve the detoxification of biologicalproducts in vivo by means of genetically modified fibroblasts implantedin a structured collagen gel with PTFE fibers.

Example 8

[0218] Use of an inducible promoter (Mx) to modulate the expression of acDNA inserted in a retroviral vector.

[0219] In the case of implants secreting a protein continuously into thecirculation, it would be desirable to be able to exercise control overthis secretion. In order to do this a retroviral vector derived from thevector M48 was constructed in which the PGK-1 promoter was excised andreplaced by a fragment of 250 bp covering the region −250 to +1 of thepromoter of the Mx gene of the mouse. This promoter comprises elementsof response to stimulation by alpha and beta interferons. In the mousethe proton Mx is expressed in response to a secretion of alpha or betainterferon. In a manner similar to that in Examples 3, 4 and 5, the cDNAcoding for mouse EPO was inserted into this vector at the BamHI site,thus generating the vector MxEPO. ψCRE cells producing recombinantecotropic retroviral particles containing the genome of the vector MxEPOwere isolated. Fibroblasts isolated from a skin biopsy performed on amouse were infected with the vector MxEPO, and the EPO secreted into thesupernatant of these cultures was measured before and after addition ofalpha interferon (1000 units per ml) in the culture medium. The basicsecretion levels of EPO were less than 0.3 units/ml/24 hours, evidencefor a low activity of the Mx promoter present in the MxEPO vector in theabsence of interferon. An increase in the secretion up to 10 fold thebasic level was measured after addition of alpha interferon. This highsecretion is maintained for at least 6 days. This observation shows thata control may be exercised over a transcriptional promoter inserted intoa retroviral vector after the integration of the latter in the genome ofthe target cell.

[0220] Implants constituted of PTFE fibers, rat tail collagen andcontaining skin fibroblasts transduced with the MxEPO vector wereprepared and implanted in syngeneic mice. In the absence of othertreatment, these animal have maintained a normal hematocrit. Some of therecipient animals were treated by the intra-peritoneal injection of 50mg of polyA-U (Boehringer) each 48 hours for two weeks in order to raisethe hematocrit.

Treatment of Genetic or Acquired Diseases

[0221] The process is applicable to the delivery to the serum of anysoluble protein or transmembrane proteins lacking their membraneimmobilization domain. However, it does not permit regulation of theserum concentrations of the foreign protein. The following applicationsare envisaged for genetic diseases: in hemophilia B, the supply offactor IX); in hemophilia A, the supply of factor VIII in the form of aprotein deleted in the B region; in beta-thalassemia, the supply ofeythropoietin capable of correcting the anemia by stimulation of fetalhemoglobin synthesis.

[0222] The supply a soluble CD4 or its derivatives coupled toimmunotoxins and immunoglobulins is conceivable in infections byretroviruses of the HIV family. More generally, any soluble anti-viralprotein could be delivered in this manner.

Production of Antibodies

[0223] The circulation of a foreign protein in the mouse induces animmune response marked by the appearance of specific antibodies as wehave observed for beta-glucuronidase, the soluble CD4 anderythropoietin. The process could thus be applied to immunise animalsagainst the still unidentified product of a cDNA isolated by reversegenetics or by other means. It should be possible to obtain polyclonalor monoclonal antibodies in this manner without necessitating the priorpreparation of the protein.

[0224] The same approach can be envisaged to achieve immunizations for atherapeutic or prophylactic purpose. It should be possible to suggestvaccinating preparations, for example, against neutralizing epitopes ofthe envelope of HIV or related viruses.

Regulation by the tet Operator/tet Repressor System

[0225] Skin fibroblasts are prepared in which two retroviral vectors areintroduced, one coding for the protein of interest placed under thecontrol of a promoter comprising the tet operator elements, the othercoding for the tet repressor or a related molecule modified in order toproduce an enhancer effect. Depending on the nature of this latterelement and that of the promoter containing the operator sequences, theaddition of tetracycline will induce an enhancement of a repression oftranscriptions. The fibroblasts transduced with these two vectors areimplanted in vivo in a neo-organ and the animals are treated withtetracycline in order to assess the induction of expression. Thereporter gene is preferably that of the mouse EPO.

Regulation by the Progresterone Receptor Modified in Order to InduceTranscription in the Presence of RU486

[0226] When its C-terminal region is cleaved the progesterone receptorbecomes capable at stimulating transcription in the presence of RU486.Skin fibroblasts are infected with two retroviral vectors, one codingfor the protein of interest expressed under the control of a promotercomprising elements of response to progesterone, the other coding for atruncated version of the progesterone receptor. As previously, thefibroblasts transduced by these two vectors are implanted in neo-organsin the mouse and the animals are treated with RU486.

1 1 1 8388 DNA mus musculus, Mo-MuLV, and other 1 tgaaagaccc cacctgtaggtttggcaagc tagcttaagt aacgccattt tgcaaggcat 60 ggaaaaatac ataactgagaatagagaagt tcagatcaag gtcaggaaca gatggaacag 120 ctgaatatgg gccaaacaggatatctgtgg taagcagttc ctgccccggc tcagggccaa 180 gaacagatgg aacagctgaatatgggccaa acaggatatc tgtggtaagc agttcctgcc 240 ccggctcagg gccaagaacagatggtcccc agatgcggtc cagccctcag cagtttctag 300 agaaccatca gatgtttccagggtgcccca aggacctgaa atgaccctgt gccttatttg 360 aactaaccaa tcagttcgcttctcgcttct gttcgcgcgc ttctgctccc cgagctcaat 420 aaaagagccc acaacccctcactcggggcg ccagtcctcc gattgactga gtcgcccggg 480 tacccgtgta tccaataaaccctcttgcag ttgcatccga cttgtggtct cgctgttcct 540 tgggagggtc tcctctgagtgattgactac ccgtcagcgg gggtctttca tttgggggct 600 cgtccgggat cgggagacccctgcccaggg accaccgacc caccaccggg aggtaagctg 660 gccagcaact tatctgtgtctgtccgattg tctagtgtct atgactgatt ttatgcgcct 720 gcgtcggtac tagttagctaactagctctg tatctggcgg acccgtggtg gaactgacga 780 gttcggaaca cccggccgcaaccctgggag acgtcccagg gacttcgggg gccgtttttg 840 tggcccgacc tgagtccaaaaatcccgatc gttttggact ctttggtgca ccccccttag 900 aggagggata tgtggttctggtaggagacg agaacctaaa acagttcccg cctccgtctg 960 aatttttgct ttcggtttgggaccgaagcc gcgccgcgcg tcttgtctgc tgcagcatcg 1020 ttctgtgttg tctctgtctgactgtgtttc tgtatttgtc tgagaatatg ggcccgcggg 1080 ccagactgtt accactcccttaagtttgac cttaggtcac tggaaagatg tcgagcggat 1140 cgctcacaac cagtcggtagatgtcaagaa gagacgttgg gttaccttct gctctgcaga 1200 atggccaacc tttaacgtcggatggccgcg agacggcacc tttaaccgag acctcatcac 1260 ccaggttaag atcaaggtcttttcacctgg cccgcatgga cacccagacc aggtccccta 1320 catcgtgacc tgggaagccttggcttttga cccccctccc tgggtcaagc cctttgtaca 1380 ccctaagcct ccgcctcctcttcctccatc cgccccgtct ctcccccttg aacctcctcg 1440 ttcgaccccg cctcgatcctccctttatcc agccctcact ccttctctag gcgccaaacc 1500 taaacctcaa gttctttctgacagtggggg gccgctcatc gacctactta cagaagaccc 1560 cccgccttat agggacccaagaccaccccc ttccgacagg gacggaaatg gtggagaagc 1620 gacccctgcg ggagaggcaccggacccctc cccaatggca tctcgcctac gtgggagacg 1680 ggagccccct gtggccgactccactacctc gcaggcattc cccctccgcg caggaggaaa 1740 cggacagctt caatactggccgttctcctc ttctgacctt tacaactgga aaaataataa 1800 cccttctttt tctgaagatccaggtaaact gacagctctg atcgagtctg ttctcatcac 1860 ccatcagccc acctgggacgactgtcagca gctgttgggg actctgctga ccggagaaga 1920 aaaacaacgg gtgctcttagaggctagaaa ggcggtgcgg ggcgatgatg ggcgccccac 1980 tcaactgccc aatgaagtcgatgccgcttt tcccctcgag aattctaccg ggtaggggag 2040 gcgcttttcc caaggcagtctggagcatgc gctttagcag ccccgctggc acttggcgct 2100 acacaagtgg cctctggcctcgcacacatt ccacatccac cggtagcgcc aaccggctcc 2160 gttctttggt ggccccttcgcgccaccttc tactcctccc ctagtcagga agttcccccc 2220 gccccgcagc tcgcgtcgtgcaggacgtga caaatggaag tagcacgtct cactagtctc 2280 gtgcagatgg acagcaccgctgagcaatgg aagcgggtag gcctttgggg cagcggccaa 2340 tagcagcttt gctccttcgctttctgggct cagaggctgg gaaggggtgg gtccgggggc 2400 gggctcaggg gcgggctcaggggcggggcg ggcgcgaagg tcctccggag cccggcattc 2460 tgcacgcttc aaaagcgcacgtctgccgcg ctgttctcct cttcttcatc tccgggcctt 2520 tcgaccggat ccggcgattagtccaatttg ttaaagacag gatatcagtg gtccaggctc 2580 tagttttgac tcaacaatatcaccagctga agcctataga gtacgagcca tagataaaat 2640 aaaagatttt atttagtctccagaaaaagg ggggaatgaa agaccccacc tgtaggtttg 2700 gcaagctagc ttaagtaacgccattttgca aggcatggaa aaatacataa ctgagaatag 2760 agaagttcag atcaaggtcaggaacagatg gaacagggtc gaccctagag aaccatcaga 2820 tgtttccagg gtgccccaaggacctgaaat gaccctgtgc cttatttgaa ctaaccaatc 2880 agttcgcttc tcgcttctgttcgcgcgctt ctgctccccg agctcaataa aagagcccac 2940 aacccctcac tcggggcgccagtcctccga ttgactgagt cgcccgggta cccgtgtatc 3000 caataaaccc tcttgcagttgcatccgact tgtggtctcg ctgttccttg ggagggtctc 3060 ctctgagtga ttgactacccgtcagcgggg gtctttcatt tatgtgtcat aaatatttct 3120 aattttaaga tagtatctccattggctttc tactttttct ttttattttt ttttgtcctc 3180 tgtctccatg tgttgttgttgttgtttgtt tgtttgtttg ttggttggtt ggttaatttt 3240 tttttaaaga tcctacactatagttcaagc tagactatta gctactctgt aacccagggt 3300 gaccttgaag tcatgggtagcctgctgttt tagccttccc acatctaaga ttacaggtat 3360 gagctatcat tttggtatattgattgattg attgattgat gtgtgtgtgt gtgattgtgt 3420 ttgtgtgtgt gattgtgtatatgtgtgtat ggttgtgtgt gattgtgtgt atgtatgttt 3480 gtgtgtgatt gtgtgtgtgtgattgtgcat gtgtgtgtgt gatgtgttag tgtatgattg 3540 tgtgtgtgtg tgtgtgtgtgtgtgtgtgtg tgtgtgtgtg tgtgtgttgt gtatatatat 3600 ttatggtagt gagaggcaacgctccggccc aggcgtcagg ttggtttttg agacagagtc 3660 tttcacttag cttgaattcttgaagacgaa agggcctcgt gatacgccta tttttatagg 3720 ttaatgtcat gataataatggtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 3780 gcggaacccc tatttgtttatttttctaaa tacattcaaa tatgtatccg ctcatgagac 3840 aataaccctg ataaatgcttcaataatatt gaaaaaggaa gagtatgagt attcaacatt 3900 tccgtgtcgc ccttattcccttttttcggg cattttgcct tcctgttttt gctcacccag 3960 aaacgctggt gaaagtaaaagatgctgaag atcagttggg tgcacgagtg ggttacatcg 4020 aactggatct caacagcggtaagatccttg agagttttcg ccccgaagaa cgttttccaa 4080 tgatgagcac ttttaaagttctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc 4140 aagagcaact cggtcgccgcatacactatt ctcagaatga cttggttgag tactcaccag 4200 tcacagaaaa gcatcttacggatggcatga cagtaagaga attatgcagt gctgccataa 4260 ccatgagtga taacactgcggccaacttac ttctgacaac gatcggagga ccgaaggagc 4320 taaccgcttt tttgcacaacatgggggatc atgtaactcg ccttgatcgt tgggaaccgg 4380 agctgaatga agccataccaaacgacgagc gtgacaccac gatgcctgca gcaatggcaa 4440 caacgttgcg caaactattaactggcgaac tacttactct agcttcccgg caacaattaa 4500 tagactggat ggaggcggataaagttgcag gaccacttct gcgctcggcc cttccggctg 4560 gctggtttat tgctgataaatctggagccg gtgagcgtgg gtctcgcggt atcattgcag 4620 cactggggcc agatggtaagccctcccgta tcgtagttat ctacacgacg gggagtcagg 4680 caactatgga tgaacgaaatagacagatcg ctgagatagg tgcctcactg attaagcatt 4740 ggtaactgtc agaccaagtttactcatata tactttagat tgatttaaaa cttcattttt 4800 aatttaaaag gatctaggtgaagatccttt ttgataatct catgaccaaa atcccttaac 4860 gtgagttttc gttccactgagcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 4920 atcctttttt tctgcgcgtaatctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 4980 tggtttgttt gccggatcaagagctaccaa ctctttttcc gaaggtaact ggcttcagca 5040 gagcgcagat accaaatactgtccttctag tgtagccgta gttaggccac cacttcaaga 5100 actctgtagc accgcctacatacctcgctc tgctaatcct gttaccagtg gctgctgcca 5160 gtggcgataa gtcgtgtcttaccgggttgg actcaagacg atagttaccg gataaggcgc 5220 agcggtcggg ctgaacggggggttcgtgca cacagcccag cttggagcga acgacctaca 5280 ccgaactgag atacctacagcgtgagctat gagaaagcgc cacgcttccc gaagggagaa 5340 aggcggacag gtatccggtaagcggcaggg tcggaacagg agagcgcacg agggagcttc 5400 cagggggaaa cgcctggtatctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 5460 gtcgattttt gtgatgctcgtcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 5520 cctttttacg gttcctggccttttcgtggc cttttgctca catgttcttt cctgcgttat 5580 cccctgattc tgtggataaccgtattaccg cctttgagtg agctgatacc gctcgccgca 5640 gccgaacgac cgagcgcagcgagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt 5700 attttctcct tacgcatctgtgcggtattt cacaccgcat atggtgcact ctcagtacaa 5760 tctgctctga tgccgcatagttaagccagt atacactccg ctatcgctac gtgactgggt 5820 catggctgcg ccccgacacccgccaacacc cgctgacgcg ccctgacggg cttgtctgct 5880 cccggcatcc gcttacagacaagctgtgac cgtctccggg agctgcatgt gtcagaggtt 5940 ttcaccgtca tcaccgaaacgcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg 6000 aagcgattca cagatgtctgcctgttcatc cgcgtccagc tcgttgagtt tctccagaag 6060 cgttaatgtc tggcttctgataaagcgggc catgttaagg gcggtttttt cctgtttggt 6120 cactgatgcc tccgtgtaagggggatttct gttcatgggg gtaatgatac cgatgaaacg 6180 agagaggatg ctcacgatacgggttactga tgatgaacat gcccggttac tggaacgttg 6240 tgagggtaaa caactggcggtatggatgcg gcgggaccag agaaaaatca ctcagggtca 6300 atgccagcgc ttcgttaatacagatgtagg tgttccacag ggtagccagc agcatcctgc 6360 gatgcagatc cggaacataatggtgcaggg cgctgacttc cgcgtttcca gactttacga 6420 aacacggaaa ccgaagaccattcatgttgt tgctcaggtc gcagacgttt tgcagcagca 6480 gtcgcttcac gttcgctcgcgtatcggtga ttcattctgc taaccagtaa ggcaaccccg 6540 ccagcctagc cgggtcctcaacgacaggag cacgatcatg cgcacccgtg gccaggaccc 6600 aacgctgccc gagatgcgccgcgtgcggct gctggagatg gcggacgcga tggatatgtt 6660 ctgccaaggg ttggtttgcgcattcacagt tctccgcaag aattgattgg ctccaattct 6720 tggagtggtg aatccgttagcgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg 6780 ctccatgcac cgcgacgcaacgcggggagg cagacaaggt atagggcggc gcctacaatc 6840 catgccaacc cgttccatgtgctcgccgag gcggcataaa tcgccgtgac gatcagcggt 6900 ccagtgatcg aagttaggctggtaagagcc gcgagcgatc cttgaagctg tccctgatgg 6960 tcgtcatcta cctgcctggacagcatggcc tgcaacgcgg gcatcccgat gccgccggaa 7020 gcgagaagaa tcataatggggaaggccatc cagcctcgcg tcgcgaacgc cagcaagacg 7080 tagcccagcg cgtcggccgccatgccggcg ataatggcct gcttctcgcc gaaacgtttg 7140 gtggcgggac cagtgacgaaggcttgagcg agggcgtgca agattccgaa taccgcaagc 7200 gacaggccga tcatcgtcgcgctccagcga aagcggtcct cgccgaaaat gacccagagc 7260 gctgccggca cctgtcctacgagttgcatg ataaagaaga cagtcataag tgcggcgacg 7320 atagtcatgc cccgcgcccaccggaaggag ctgactgggt tgaaggctct caagggcatc 7380 ggtcgacgct ctcccttatgcgactcctgc attaggaagc agcccagtag taggttgagg 7440 ccgttgagca ccgccgccgcaaggaatggt gcatgcaagg agatggcgcc caacagtccc 7500 ccggccacgg ggcctgccaccatacccacg ccgaaacaag cgctcatgag cccgaagtgg 7560 cgagcccgat cttccccatcggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 7620 gcgccggtga tgccggccacgatgcgtccg gcgtagagcg ccacaggacg ggtgtggtcg 7680 ccatgatcgc gtagtcgatagtggctccaa gtagcgaagc gagcaggact gggcggcggc 7740 caaagcggtc ggacagtgctccgagaacgg gtgcgcatag aaattgcatc aacgcatata 7800 gcgctagcag cacgccatagtgactggcga tgctgtcgga atggacgata tcccgcaaga 7860 ggcccggcag taccggcataaccaagccta tgcctacagc atccagggtg acggtgccga 7920 ggatgacgat gagcgcattgttagatttca tacacggtgc ctgactgcgt tagcaattta 7980 actgtgataa actaccgcattaaagctttg cttaggagtt tcctaataca tcccaaactc 8040 aaatatataa gcatttgacttgttctatgc cctaggggga ggggggaagc taagccagct 8100 ttttttaaca tttaaaatgttaattccatt ttaaatgcac agatgttttt atttcataag 8160 ggtttcaatg tgcatgaatgtcgcaatatc ctgttaccaa agctagtata aataaaaata 8220 gataaacgtg gaaattacttagagtttctg tcattaacgt ttccttcctc agttgacaac 8280 ataaatgcgc tgctgagaagccagtttgca tctgtcagga tcaatttcca ttatgccagt 8340 catattaatt actagtcaattagttgattt ttgacatata catgtgaa 8388

1. Implant characterized in that it comprises: a biocompatible supportpermitting the biological anchoring of cells, cells having the capacityto express and secrete naturally or after recombination a definedsubstance, for example a substance having therapeutic value; and aconstituent capable of inducing and/or promoting the gelation of saidcells.
 2. Implant according to claim 1, characterized in that thebiocompatible support includes at least me of the elements selected fromthe group comprising PTFE or a support of biological origin.
 3. Implantaccording to claim 1, characterized in that the biocompatible support isa support of biological origin of the type resorbable in vivo at leastpartially.
 4. Implant according to any one of the claims 1 to 3,characterized in that the biocompatible support is a calcium-based, inparticular a calcium carbonate-based, support, preferably it is coral.5. Implant according to claim 4, characterized in that the biocompatiblesupport is high-porosity coral.
 6. Implant according to claim 3 or claim5, characterized in that the high-porosity coral is a spherical coral.7. Implant according to any one of the claims 1 to 6, characterized inthat the constituent capable of inducing and/or promoting the gelationof the cells is collagen, in particular type I collagen, preferably at aconcentration of the order of 1.5 mg /ml.
 8. Implant according to claim1 or claim 3, characterized in that the biocompatible support isselected from: cross-linked collagen, in particular in the form offibers or sponges, bone powder, carbohydrate-based polymers such asdextran or hyaluronic acid.
 9. Implant according to any one of theclaims 1 to 8, characterized in that the constituent capable of inducingand/or promoting the gelation of the cells is selected from supportsbased on: uncross-linked collagen, alginates.
 10. Implant according toany one of the claims 1 to 9, characterized in that the cells arerecombinant cells having the capacity to be tolerated immunologically byan organism to which they are administered, modified by a nucleotidesequence coding for a defined polypeptide.
 11. Implant according toclaim 10, characterized in that the recombinant cells are fibroblasts,in particular skin fibroblasts.
 12. Implant according to any one of theclaims 1 to 11, characterized in that said cells are recombinant cellsmodified by a retroviral vector comprising a proviral DNA sequencemodified in a manner such that: the gag p and env genes of the proviralDNA have been deleted at least in part in order to produce a proviralDNA incapable of replicating this DNA being in addition incapable ofrecombining to form a wild-type virus, the LTR sequence contains adeletion in the sequence U3 such that transcription of the mRNA that itcontrols is reduced significantly for example at least 10-fold, and therecombinant retroviral vector comprises, in addition, an exogenousnucleotide sequence under the control of a promoter for example anexogenous, constitutive or inducible promoter.
 13. Implant according toclaim 12, characterized in that the proviral DNA of the vector isderived from the MuLV retrovirus.
 14. Implant according to claim 11 orclaim 12, characterized in that the sequences of the pol and env genesof the proviral DNA are entirely deleted.
 15. Implant according to anyone of the claims 11 to 13, characterized in that the U3 region of theLTR3′ fragment of the proviral DNA is deleted at the level of nucleotide2797 of FIG.
 1. 16. Implant according to any one of the claims 12 to 15,characterized in that the exogenous nucleotide sequence is under thecontrol of the mouse PGK-1 promoter or the human PGK-1 promoter,optionally lacking a “TATA box”.
 17. Implant according to any one of theclaims 12 to 16, characterized in that the proviral sequence upstreamfrom the exogenous promoter is the proviral nucleotide sequence situatedbetween nucleotides 1 and about 1500 of the sequence shorn in FIG. 1.18. Implant according to any one of the claims 12 to 17, characterizedin that the retroviral vector is the vector pM48 show in FIG. 2,modified by the insertion of the exogenous nucleotide sequence at theBamHI site.
 19. Implant according to any one of the claims 1 to 10 or 12to 18, characterized in that the recombinant cells are tumor cells. 20.Implant according to any one of the claims 1 to 19, characterized inthat the recombinant cells are modified by a vector containing one ormore exogenous nucleotide sequences coding for an antigen or anantigenic determinant or coding for a polypeptide or glycoproteinsoluble in the serum, for example a polypeptide or a glycoprotein oftherapeutic interest, in particular a hormone, a structural protein orglycoprotein or a metabolic protein or glycoprotein or a viral proteinor glycoprotein or a protein having the characteristics of an antibodyor an antibody fragment.
 21. Implant according to any one of the claims1 to 20, characterized in that it contains in addition one or moreangiogenic factors, in particular bFGF.
 22. Implant according to any oneof the claims 1 to 21, characterized in that it contains heparin or aheparin derivative.
 23. Implant according to any one of the claims 1 to22, characterized in that it contains from 10⁶ to 10⁹, and preferablyfrom 5×10⁶ to 10⁷ recombinant cells.
 24. Use of an implant according toany one of the claims 1 to 23, in a permanent or temporary fashion, forthe implantation in man or animals.
 25. Use of an implant according toany one of the claims 1 to 23: either for the treatment of geneticdiseases, in particular for the treatment of diseases of lysosomaloverload, hemophilia A or hemophilia B, beta-thalassemia, the erogenousnucleotide sequence contained in the recombinant cells correspondingrespectively to those which code for beta-glucuronidase, for the factorVIII factor IX or erythropoietin, or for an active part of thesesequences; or for the treatment of acquired diseases, for example forthe treatment of viral diseases in particular for the treatment of aninfection due to the HIV retrovirus, for example by the expression andsecretion into the serum of soluble CD4 molecules or a solubleanti-viral protein; or for the treatment of tumors, the exogenousnucleotide sequence contained in the recombinant cells coding for asubstance capable of promoting or enhancing the immune response againstthe cells of the tumors.
 26. Composition characterized in that itcontains an implant according to any one of the claims 1 to 23 with oneor more substances, in particular an antigen or an adjuvant.
 27. Methodof treatment of genetic diseases, acquired diseases or tumors, saidmethod comprising the introduction into man or animals of an implantaccording to any one of the claims 1 to 23 for a period of timesufficiently long to allow the cells included in said implant to producein vivo a peptide, a protein or a glycoprotein having a therapeuticeffect on the disease to be treated.
 28. Method according to claim 7,characterized in that said implant is introduced in the peritonealcavity, the peri-renal space or the skin of the patient to be treated.29. Method of preparation of an implant according to any one of theclaims 1 to 23, said method comprising the steps of: placing of thebiocompatible support in contact with said cells and a constituentcapable of inducing and/or promoting their gelation; incubation of thepreparation obtained in the previous step in order to obtain thegelation of said constituents; culture of the cells thus obtained underconditions allowing them to bind to the gelled constituents, andrecovery of the implant thus obtained.
 30. Method according to claim 29,characterized in that the biocompatible support is placed in contactwith cells previously incorporated into a solution of collagen. 31.Method according to claim 29 or 30, characterized in that thebiocompatible support is constituted of PTFE fibers or coral powder,previously treated with a solution of collagen or a growth factor. 32.Recombinant retroviral vector characterized in that it comprises: aproviral DNA sequence modified in a manner such that: the gag, pol andenv genes of the proviral DNA have been deleted at least in part inorder to produce a proviral DNA incapable of replication, this DNA beingin addition unable to recombine for form a wild-type virus, the LTRsequence bears a deletion in the U3 sequence such that transcription ofmRNA that it controls is reduced significantly, for sample by at least10 fold, and the recombinant retroviral vector comprising in addition anexogenous nucleotide sequence under the control of a promoter forexample an exogenous, inducible or constitutive promoter.
 33. Retroviralvector according to claim 32, characterized in that the modifiedproviral DNA sequence, the exogenous nucleotide sequence and theexogenous promoter are borne by a plasmid.
 34. Retroviral vectoraccording to claim 1 or claim 33, characterized in that the proviral DNAis derived from the MuLV retrovirus.
 35. Retroviral vector according toany one at the claims 32 to 34, characterized in that the sequences forthe pol and env genes of the proviral DNA are entirely deleted. 36.Retroviral vector according to any one of the claims 32 to 35,characterized in that the U3 region of the LTR3′ fragment is deleted atthe level of nucleotide 2797 of FIG.
 1. 37. Retroviral vector accordingto any one of the claims 32 to 36, characterized in that the exogenousnucleotide sequence is under the control of the mouse PGK-1 promoter orthe human PGK-1 promoter, optionally lacking a “TATA box”. 38.Retroviral vector according to any one of the claims 32 to 36,characterized in that the proviral sequence upstream from the exogenouspromoter is the proviral nucleotide sequence situated between thenucleotides 1 and about 1500 of the sequence shown in FIG.
 1. 39.Retroviral vector according to claim 37, characterized in that theexogenous nucleotide sequence is inserted at the BamHI site downstreamfrom the exogenous constitutive PGK-1 promoter.
 40. Retroviral vectoraccording to any one of the claims 32 and 39, characterized in that itis the pM48 vector shown in FIG. 2, modified by the insertion of theexogenous nucleotide sequence at the BamHI site.
 41. Retroviral vectoraccording to any one of the claims 32 to 40, characterized in that itcontains at the BamHI site downstream from the exogenous constitutivepromoter a BamHI fragment of the gene of beta-galactosidase. 42.Retroviral vector acceding to any one of the claim 32 to 41,characterized in that it contains in addition upstream from theexogenous constitutive promoter an enhancer sequence.
 43. Recombinantcells characterized in that they are cells having the capacity to betolerated immunologically by the organism to which they areadministered, modified by a retroviral vector according to any one ofthe claims 32 to
 42. 44. Recombinant cells according to claim 43,characterized in that they are recombinant fibroblasts, in particularskin fibroblasts.
 45. Recombinant cells according to claim 43,characterized in that they are tumor cells modified by a retroviralvector according to any one of the claims 32 to
 42. 46. Recombinantcells according to any one of the claims 43 to 45, characterized in thatthe exogenous nucleotide sequence which they contain codes for a proteinwhose expression is desired, in particular a protein soluble in theserum.
 47. Recombinant cells according to any one of the claims 43 to46, characterized in that the exogenous nucleotide sequence which theycontain codes for beta-glucuronidase.
 48. Use of the recombinant cellsaccording to any one of the claims 43 to 47 for the treatment of adisease capable of being corrected by the expression and secretion intothe serum of a patient of the exogenous nucleotide sequence contained inthese cells.
 49. Use of the recombinant cells according to any one ofthe claims 43 to 47 for the treatment of genetic diseases, in particularfor the treatment of diseases of lysosomal overload, hemophilia A orhemophilia B, beta-thalassemia, the exogenous nucleotide sequencecontained in the recombinant cells corresponding respectively to thosewhich code for beta-glucuronidase for the factor VIII, factor IX orerythropoietin or for an active part of these sequences.
 50. Use of therecombinant cells according to any one of the claims 43 to 47 for thetreatment of acquired diseases, for Ocala for the treatment of viraldiseases, in particular for the treatment of an infection due to the HIVretrovirus for example by the expression and secretion into the serum ofsoluble CD4 molecules or of a soluble anti-viral protein.
 51. Use of therecombinant cells according to any one of the claims 43 to 47 for thepreparation of antibodies against the expression product of theexogenous nucleotide sequence contained in the recombinant cells. 52.Use of the recombinant cells according to any one o the claims 43 to 47for the treatment of tumors, the exogenous nucleotide sequence containedin the recombinant cells coding for a substance capable of promoting orenhancing the immune response against the cells of the tumor. 53.Recombinant cells according to claim 52 such as obtained byrecombination of tumor cells with a retroviral vector according to anyone of the claims 32 to
 42. 54. Kit for the preparation of an implant toachieve the in vivo expression and secretion by cells of a substance toproduce a desired therapeutic effect, said kit containing: abiocompatible support making possible the biological anchoring of saidcells; and a constituent capable of inducing and/or promoting thegelation of said cells.
 55. Kit according to claim 54, characterized inthat the biocompatible support comprises at least one of the elementsselected from the group including PTFE or a support of biologicalorigin, in particular a calcium-based, in particular a calciumcarbonate-based, support of biological origin, preferably coral.
 56. Kitaccording to claim 54 or 55, characterized in that the constituentcapable of inducing and/or promoting the gelation of the cells iscollagen, in particular type I collagen, preferably at a concentrationof the order of 1.5 mg/ml.
 57. Kit according to claim 54, characterizedin that it contains a DNA comprising a sequence coding for the substanceexpressed and secreted by said cells.
 58. Kit according to claim 57,characterized in that it contains a retroviral vector according to anyone of the claims 32 to
 42. 59. Kit according to any one of the claims54 to 58, characterized in that it contains cells having the capacity toexpress and secrete naturally CT after recombination a defined substancefor example a substance having a therapeutic value.
 60. Kit according toclaim 59, characterized in that the cells are recombinant cells aaccording to anyone of the claims 43 to 47.